Flow injection system for on-line monitoring of fumaric acid in biological processes

Abstract

A flow injection system has been developed for monitoring fumaric acid in biological processes by using fumarase and malic dehydrogenase immobilized on VA-Epoxy Biosynth E3-carrier. An immobilized fumarase and malic dehydrogenase reactor had a good operational lifetime (up to 18 h) and storage stability (up to 10 days). The flow injection analysis (FIA) system with the immobilized enzymes cartridge was characterized according to the factors affecting the activity of the immobilized enzymes, such as pH, temperature, sample matrix, etc. The optimal pH value of the immobilized enzymes was slightly shifted in the alkaline range, i.e. 9.0. Some components such as 1 mM imidazole, 5 mM sodium azide and 2 g l−1 semicarbazide in a carrier buffer solution had significant inhibitory or activating effects on the response of the FIA system. But many components in the sample solution had no significant effects on the activity of the immobilized enzymes, except in the case of the 2 g l−1 acetate sample. The linear concentration ranges of fumaric acid were investigated by changing the volumetric packing ratio of two immobilized enzymes in a cartridge. The cartridge with a 1:1 packing ratio had a linear concentration range of 0.05–0.3 g l−1 with a correlation coefficient (r2) of 0.996, while the enzyme cartridge with a 1:2 packing ratio (or 1:4 packing ratio) had 0.1–0.6 g l−1 with r2=0.998 (or 0.3–1.2 g l−1 with r2=0.996). The FIA system utilizing the enzyme cartridge has been applied in order to monitor on-line the concentrations of fumaric acid in a few biological processes, i.e. a continuously stirred reactor and a cultivation process of Rhizopus oryzae Korean Collection for Type Cultures 6946.